This invention relates to a method of protein analysis. More particularly it relates to a method of analyzing proteins to obtain information which can be used to determine the structure of the protein, namely sequence of the amino acids making up the protein.
In the past, protein sequencing has been carried out by techniques such as those involving the sequential removal of amino acids from one end of the protein and identifying each removed amino acid in turn. Other techniques have relied on the genetic code, using the base sequence of the gene coding for the protein. Both these techniques are slow, complex and difficult.
More recent techniques have attempted to obtain amino acid sequence information using mass spectrometry, typically using fast atom bombardment to ionize the sample. In fast atom bombardment, a sample dissolved in a liquid is bombarded with atoms or ions. Charged molecules resulting from this process are directed into the mass spectrometer and detected. An example of this technique is described in the text entitled xe2x80x9cMacro Molecular Sequencing and Synthesis Selected Methods and Applicationsxe2x80x9d, 1988, published by Alan R. Liss, Inc., specifically at pages 83 to 99 in an article in such text entitled xe2x80x9cMass Spectrometry in Bio-Pharmaceutical Researchxe2x80x9d by Steven A. Carr et al.
A difficulty with the technique using mass spectrometry is that when complex protein molecules are fragmented, analysis of the daughter or fragment ions has been extremely difficult. As noted by Carr et al at page 86 of the above identified text, a Y-B analysis technique can be used to determine sequence information. However the analysis is complex, slow and difficult, and so far as the applicant is aware has never been commercially used.
According to the invention an improved method of analyzing proteins is provided, utilizing ion evaporation followed by tandem mass spectrometry. The method of the invention provides tryptic fragments which are predominantly doubly charged, one charge being located at each end of the fragment. Such fragments are then further fragmented into two singly charged daughter fragments or daughter ions in the tandem mass spectrometer, providing information which can be much more readily used to obtain the sequence of the amino acids in the protein.
In its broadest aspect the invention provides a method of analyzing a protein comprising the steps of:
(1) adding trypsin to said protein to form a liquid phase mixture of trypsin and said protein,
(2) optionally reducing the disulfide linkages and alkylating the resulting sulfhydryl groups of said protein either before or after said step (1),
(3) allowing the trypsin to digest said protein long enough to cleave said protein into tryptic fragments in said liquid phase,
(4) ionizing a portion of the digested mixture by ion evaporation to produce gas phase ions of said tryptic fragments from said liquid phase, said gas phase ions being predominantly doubly charged with one charge at each end of said doubly charged ions,
(5) and analyzing said gas phase ions of said tryptic fragments by sequentially selecting therefrom ions of a desired mass to charge ratio in a first mass analyzer, fragmenting such selected ions by collision in a second mass analyzer to produce daughter ions, and then analyzing said daughter ions in a third mass analyzer.
Further objects and advantages of the invention will appear from the following description, taken together with the accompanying drawings.